Prematurity/preterm birth is the leading cause of neonatal death in the U.S. and in California. During an average day in California, 149 babies are born preterm. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. Finally, prematurity and fetal growth restriction are many times the result of obstetric diseases, such as pregnancy-induced hypertension and seizures, which carry high rates of maternal morbidity and mortality. All the above-mentioned diseases result from abnormal development and function of the placenta, which is a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cell type which carries out major placental functions such as establishing blood supply from the mother to the fetus. This application proposes the placenta as a novel target for stem cell therapy and seeks generation of trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta. This research will lead to identification of specific trophoblast markers, which can then be developed into diagnostic tools to use in prenatal screening for placental function and fetal well-being. Most importantly, it will lead to development of trophoblast stem cell-based therapeutics for in-utero intervention in cases of fetal growth restriction, preterm labor, and pre-eclampsia. Prevention of these complications will substantially decrease both neonatal and maternal morbidity and mortality; in addition, treatment of growth restriction in the fetus will in turn decrease cardiovascular disease and diabetes in later life. Thus funding this grant would be a small investment towards substantially improving the health of future generations and alleviating the financial burden caused by premature birth and its complications in California.

Statement of Benefit to California:

The goal of every pregnant mother is to have a healthy baby. Unfortunately, pregnancy can be complicated by many diseases, which affect the placenta, the lifeline of the baby. When the placenta develops abnormally or malfunctions, babies are born small and/or premature. This leads to many complications in the perinatal period, including bleeding and abnormal development of the brain, blindness, and gastrointestinal problems. These complications usually require long stays in neonatal intensive care units, with an average per patient cost of over $20,000, compared to $1,300 for a normal weight term baby in the regular newborn nursery. Even when the small premature baby survives the neonatal period, s/he will face an increased risk of complications as a teenager and later an adult, including cardiovascular disease and diabetes, adding to the already over-burdened healthcare system. But what if we could prevent all these problems before they started, in utero, by treating the placenta? The research proposed in this application could make that possibility a reality, by developing trophoblast stem cells. There is very little known about trophoblast stem cells, because the majority of stem cell research today is focused on embryonic stem cells that become the embryo and give rise to adult organs, like the brain, heart, and pancreas. Virtually nothing is known about the “other” stem cells in the human blastocyst, the trophoblast shell that becomes the placenta. This research would start a whole new direction in the field of stem cell research. It would lead to a greater understanding of placental development and diseases, which many mothers and babies face every day. But most importantly, it would benefit the state of California by leading to therapies aimed at preventing pre-term birth and fetal growth restriction. This will not only improve the health of the future generation of Californians, it would save the state over $200 million annually in neonatal care. Aside from direct contributions towards improving the health of California’s population, funding this grant will contribute to jump-starting careers of not just one, but two physician-scientists, which are extremely rare in the field of perinatal medicine, not just in California, but world-wide. Funding this grant would greatly contribute to the development of two leaders in this field and, through their interactions with other physicians-in-training, lead to attraction of more trainees to research careers in perinatal medicine.

Progress Report:

Year 1

Prematurity/preterm birth is the leading cause of perinatal morbidity and mortality both in the U.S. and in California. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. The majority of these perinatal complications result from abnormal development and function of the placenta, a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cells which carry out major placental functions such as establishing blood supply from the mother to the fetus. In this application, we proposed the placenta as a novel target for stem cell therapy and sought to generate human trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta.
During the first year of this grant period, we have focused on characterization of a protein, which is promising as a marker for trophoblast proliferation and regeneration. We have confirmed that this protein, named p63, in fact plays a major role in keeping placenta-derived trophoblast cells regenerating in a culture dish. We have also discovered that this protein is expressed early after induction of human embryonic stem cell differentiation into trophoblast. We are currently testing the effect of up- and down-regulation of this protein in both human placenta-derived and human embryonic stem cell-derived trophoblast. We hope that by modifying this single protein, we will be able to maintain human trophoblast in culture. This would be a major step towards identification and generation of true human trophoblast stem cells.

Year 2

Prematurity/preterm birth is the leading cause of perinatal morbidity and mortality both in the U.S. and in California. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. The majority of these perinatal complications result from abnormal development and function of the placenta, a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cells which carry out major placental functions such as establishing blood supply from the mother to the fetus. In this application, we proposed the placenta as a novel target for stem cell therapy and sought to generate human trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta.
During the past year, we have made significant progress in two areas. First, we have learned that p63, a protein we had previously identified as a potential marker of trophoblast proliferation and regeneration, in fact regulates specific properties associated with stemness in trophoblast. We are currently attempting to prolong the lifespan of primary trophoblast in culture by introducing p63, along with other genes, into these cells. In addition, p63 is also expressed early after induction of human embryonic stem cell differentiation into trophoblast, and is maintained under low oxygen conditions, which are known to maintain trophoblast proliferation. We are currently testing the effect of down-regulation of this protein on trophoblast induction of human embryonic stem cells. We believe that this single protein plays a major role in developing and maintaining proliferating trophoblast in culture.
In order to discover additional markers for trophoblast proliferation and regeneration, we have collected samples from placentas of various gestational ages. It is known that trophoblast in early placenta (especially in first trimester) are more proliferative than those in later pregnancy (i.e. third trimester). By looking at total gene expression in placentas across gestation, we hope to identify more markers like p63, which play a role in trophoblast stemness. In addition to collection of placental samples, which include a mixture of trophoblast and other cell types, we have also worked on optimizing isolation of a pure population of trophoblast from placentas of different gestational ages. Aside from looking at gene expression, we are also optimizing culture conditions (including determining the best oxygen tension) for these cells.

Year 3

Prematurity/preterm birth is the leading cause of perinatal morbidity and mortality both in the U.S. and in California. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. The majority of these perinatal complications result from abnormal development and function of the placenta, a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cells which carry out major placental functions such as establishing blood supply from the mother to the fetus. In this application, we proposed the placenta as a novel target for stem cell therapy and sought to generate human trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta.
During the past year, we have made significant progress in characterization of p63, a protein we had previously identified as a potential marker of proliferative trophoblast. We now know that this protein: 1) is upregulated in low oxygen, conditions that promote trophoblast proliferation; and 2) it regulates the trophoblast cell cycle at a specific point. When we forcibly increase its production in trophoblast, p63 keeps the cells from differentiating into hCG-secreting syncytiotrophoblast; however, by itself, it is unable to keep human trophoblast cells proliferative in culture. In addition, we now have evidence that p63 plays a major role in differentiation of human embryonic stem cells (hESC) towards the trophoblast lineage. We are now testing culture conditions under which p63-expressing hESC-derived trophoblast can be maintained, and not progress to terminally differentiated trophoblast.
In order to discover additional markers for trophoblast proliferation and regeneration, we have collected samples from placentas of various gestational ages and subjected these to gene expression analysis. It is known that trophoblast in early placenta (especially in first trimester) are more proliferative than those later in pregnancy (i.e. third trimester). By looking at total gene expression in placentas across gestation, we have now identified several other markers like p63, which have a high potential to play a role in trophoblast “stemness.” Over the next year, we will be evaluating the localization of these gene products in the placenta and evaluating their function in trophoblast differentiation.

Year 4

Prematurity/preterm birth is the leading cause of perinatal morbidity and mortality both in the U.S. and in California. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. The majority of these perinatal complications result from abnormal development and function of the placenta, a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cells which carry out major placental functions such as establishing blood supply from the mother to the fetus. In this application, we proposed the placenta as a novel target for stem cell therapy and sought to generate human trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta.
During the past year, we have completed our characterization of p63, a protein we had previously identified as a potential marker of “cytotrophoblast,” the proliferative trophoblast “stem” cell in the placenta. We have a manuscript in review describing its role in maintaining the cytotrophoblast cell state in the placenta, as well as its role in inducing the trophoblast lineage in human embryonic stem cells (hESCs). In addition, we have established a protocol for differentiating hESCs first into a pure culture of cytotrophoblast, then differentiating them further into the two distinct functional, hormone-secreting trophoblast subtypes. Finally, we have learned that hypoxia both accelerates differentiation of hESCs into trophoblast, and also favors differentiation into the invasive type of trophoblast. We are now attempting to establish “defined” culture conditions, which can reproducibly differentiate hESCs into each specific trophoblast subtype.
We have also completed analysis of gene expression changes in different gestation placental samples, isolated cytotrophoblast, and differentiated cytotrophoblast, and identified multiple additional genes with potential role for maintaining the proliferative cytotrophoblast stem cell niche. A few of these have previously been shown to play a role in maintaining trophoblast stem cells in the mouse, but most appear to be specific to the human placenta. We are currently focusing on the genes involved in signaling (either transcription factors which control expression of other genes in the nucleus of the cell, or kinases which control signals in the cytoplasm of the cell) in order to determine how proliferation and the stem cell state is regulated in the human trophoblast. Over the next year, we will be evaluating the localization of these gene products in the human placenta and evaluating their function in trophoblast differentiation.
Finally, we are proceeding to address how the cytotrophoblast stem cell differentiates further into functional trophoblast. We are in process of isolating the different cell types in the placenta, then plan to characterize their gene expression, as we did with the placental and cytotrophoblast samples above, in order to identify genes involved in regulating differentiation decisions.

Year 5

Prematurity/preterm birth is the leading cause of perinatal morbidity and mortality both in the U.S. and in California. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. The majority of these perinatal complications result from abnormal development and function of the placenta, a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cells which carry out major placental functions such as establishing blood supply from the mother to the fetus. In this application, we proposed the placenta as a novel target for stem cell therapy and sought to generate human trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta.
During the past year, we have completed our characterization of p63, a protein we had previously identified as a potential marker of “cytotrophoblast,” the proliferative trophoblast “stem” cell in the placenta. We published a manuscript describing its role in maintaining the cytotrophoblast stem cell state in the placenta, as well as its role in inducing the trophoblast lineage in human embryonic stem cells (hESCs). As part of the same study and publication, we also showed that, when compared to many different types of human cells and tissues, hESC-derived cytotrophoblast most closely resemble trophoblast isolated from the placenta. This was an important milestone, as a recent publication had challenged the validity of the hESC-derived cells as bona fide trophoblast.
In the previous year, we had established a protocol for differentiating hESCs first into a pure culture of cytotrophoblast stem cells, then differentiating them further into the two distinct functional, hormone-secreting trophoblast subtypes. This past year, we have fine-tuned the culture conditions further, learning that both oxygen tension and the extracellular matrix (ECM)—the material the cells “sit on” in culture--may direct them further into each of the trophoblast subtypes. We are currently testing a combination of oxygen tension and ECM materials in order to optimize differentiation of the cytotrophoblast stem cells into either of the two main trophoblast subtypes: “villous” trophoblast which secretes the pregnancy hormone hCG, and “extravillous” trophoblast which invades the uterine tissue in order to gain access to maternal blood for fetal growth.
The previous year, we had started to analyze gene expression changes in placental samples from different gestational ages, as well as isolated cytotrophoblast, and differentiated trophoblast. We identified multiple additional genes with potential role for maintaining the proliferative cytotrophoblast stem cell niche. We performed extensive analysis on these data during the past year and found that very few have previously been shown to play a role in maintaining trophoblast stem cells in the mouse. Most appear to be specific to the human placenta. We presented this work at an international conference on the placenta this past year and are currently working on a new manuscript detailing these findings. In addition, we have been evaluating the localization and function of a handful of these genes, focusing mostly on transcription factors, because these factors tend to act as “master switches,” regulating cell behavior. Overall, these data have given us extensive insight into how the human placental develops over time. In the near future, we plan to evaluate these same genes in placentas of abnormal pregnancies, including those complicated by diseases leading to preterm birth. By understanding mechanisms of placental cell differentiation, we will be able to identify ways of targeting the placenta in disease and hopefully decrease the need for preterm delivery.

Year 6

Prematurity/preterm birth remains the leading cause of perinatal morbidity and mortality both in the U.S. and in California. These babies are at increased risk for long-term disabilities, including cerebral palsy, gastrointestinal problems, and vision and hearing loss. Many premature babies also suffer from low birth weight, which not only increases complications in the perinatal period, but also leads to increased cardiovascular disease and diabetes in adulthood. The majority of these perinatal complications result from abnormal development and function of the placenta, a transient organ that forms the interface between mother and baby. Trophoblasts are the primary cells which carry out major placental functions such as establishing blood supply from the mother to the fetus. In this application, we proposed the placenta as a novel target for stem cell therapy and sought to generate human trophoblast stem (TS) cells, which give rise to all subtypes of trophoblasts in the placenta.
During the last year of this grant, we had established a protocol for differentiating human embryonic stem cells (hESCs) first into a pure culture of TS cells, then differentiating them further into the two distinct functional, hormone-secreting trophoblast subtypes. During the past few months, we tried several conditions in order to maintain the hESC-derived TS cells in their stem cell state. We learned that while low oxygen keeps them from differentiating further, it does not maintain their ability to divide. For the latter, we will likely need a combination of low oxygen, appropriate matrix for the cells to "sit" on, and other chemicals in the culture medium. We recently wrote and submitted a grant to NIH to pursue this line of questioning.
The previous year, we had started to analyze gene expression changes in placental samples from different gestational ages, as well as isolated cytotrophoblast, and differentiated trophoblast. We identified multiple additional genes with potential role for maintaining the proliferative cytotrophoblast stem cell niche. We performed extensive analysis on these data during the past year and found that very few have previously been shown to play a role in maintaining trophoblast stem cells in the mouse. Most appear to be specific to the human placenta. We are currently working on a new manuscript detailing these findings. In addition, we evaluated the localization and function of a few of these genes, focusing mostly on transcription factors, because these factors tend to act as “master switches,” regulating cell behavior. We plan to expand on these findings in the near future; to that end, we have incorporated these findings in the above-mentioned grant to NIH, and are hoping to be able to follow up these findings using this new source of funding.
In summary, thanks to this grant, we have identified the human TS cell niche in the placenta, have developed a protocol for their derivation from hESCs, and begun to unravel the mechanism by which they contribute to the development and function of this important organ. In the near future, we plan to evaluate this stem cell niche in placentas of abnormal pregnancies, including those complicated by diseases leading to preterm birth. By understanding mechanisms of placental cell differentiation, we will be able to identify ways of targeting the placenta in disease and hopefully decrease the need for preterm delivery.